Cellular Fatty Acid Methyl Esters Research Articles

Plant growth-enhancing traits of rhizobacteria isolated from brinjal, okra, and leaf mustard

Exploring new plant-associated rhizobacteria and reintroducing them to the crop-soil environment is among the strategies to reverse the declining quality of the agricultural soil environment. A total of 340 isolates were recovered from the rhizospheres of okra (Abelmoschus esculentus), leaf mustard (Brassica juncea), and brinjal (Solanum melongena). From among the total, 155 isolates (45.59%) were diazotrophs, and 122 (35.88%) and 127 (37.35%) were isolates with the solubilising activity of phosphate and potassium, respectively. Thirteen of the most promising isolates were identified by gas chromatography for their cellular fatty acid methyl esters: Escherichia vulneris (TC19), Klebsiella pneumoniae (BA5, SA19, SB22), Pantoea ananatis (TC22), Pseudomonas aeruginosa (BA46), Pseudomonas putida (BA29, BA37, SC5, SC14), Salmonella bongori (BC17), Salmonella enterica (BB2), and Shigella dysenteriae (BA16). Three Pseudomonas strains viz. P. aeruginosa BA46 and P. putida BA37 and SC5 were selected after enhancing root elongation and vigour of lettuce seedlings. The American lettuce growth performance in the non-circulating hydroponic system was established. The inoculation with rhizobacteria strains, SC5 and BA46, stimulated shoot and root biomass over uninoculated control plants. The inoculation of lettuce plants with isolates BA37, BA46 and SC5, produced significantly longer roots compared to uninoculated control plants. This study indicates the potential use of the strains BA46 and SC5 as candidates for the formulation and production of hydroponic nutrient solution fortified with beneficial bacteria.

Isolation and Characterization of Capnocytophaga bilenii sp. nov., a Novel Capnocytophaga Species Detected in a Gingivitis Subject.

Capnocytophaga species are commensal gliding bacteria that are found in human and animal oral microbiota and are involved in several inflammatory diseases, both in immunocompromised and immunocompetent subjects. This study contributes to increased knowledge of this genus by characterizing a novel species isolated from a dental plaque sample in a male with gingivitis. We investigated morphological and chemotaxonomic characteristics using different growth conditions, temperature, and pH. Cellular fatty acid methyl ester (FAME) analysis was employed with gas chromatography/mass spectrometry (GC/MS). Phylogenetic analysis based on 16S rRNA, orthologous average nucleotide identity (OrthoANI), and digital DNA–DNA hybridization (dDDH) relatedness were performed. The Marseille-Q4570T strain was found to be a facultative aerobic, Gram-negative, elongated, round-tipped bacterium that grew at 25–56 °C and tolerated a pH of 5.5 to 8.5 and an NaCl content ranging from 5 to 15 g/L. The most abundant fatty acid was the branched structure 13-methyl-tetradecanoic acid (76%), followed by hexadecanoic acid (6%) and 3-hydroxy-15-methyl-hexadecanoic acid (4%). A 16S rDNA-based similarity analysis showed that the Marseille-Q4570T strain was closely related to Capnocytophaga leadbetteri strain AHN8855T (97.24% sequence identity). The OrthoANI and dDDH values between these two strains were, respectively, 76.81% and 25.6%. Therefore, we conclude that the Marseille-Q4570T strain represents a novel species of the genus Capnocytophaga, for which the name Capnocytophaga bilenii sp. nov. is proposed (=CSUR Q4570).

Isolation and Description of Catonella massiliensis sp. nov., a Novel Catonella Species, Isolated from a Stable Periodontitis Subject.

The genus Catonella currently counts a unique species, C. morbi, isolated from periodontal pockets and associated with periodontitis and endodontic infections. This study contributed to the taxonomical and clinical knowledge of this genus by describing a novel species isolated from a saliva sample from a man in clinical gingival health following successful treatment of periodontitis. Morphological and chemotaxonomic characteristics were investigated using different growth conditions, pH, and temperature. Cellular fatty acid methyl ester (FAME) analysis was conducted by gas chromatography/mass spectrometry (GC/MS). Phylogenetic analysis based on 16S rRNA, orthologous average nucleotide identity (OrthoANI), and digital DNA-DNA hybridization (dDDH) relatedness were performed. Strain Marseille-Q4567T was found to be an anaerobic and non-spore-forming rod-shaped bacterium that grew at 28–41.5 °C (optimum 37 °C), pH 6.5–8.5 (optimum pH 7.5), and 5–10 g/L of NaCl (optimum 5 g/L). The predominant cellular fatty acid was C16:0 (64.2%), followed by unsaturated structures C18:1n9 (12.5%) and C18:2n6 (7.8%). Based on 16S rRNA sequence comparison, the closest phylogenetic neighbor was C. morbi ATCC 51271T (98.23% similarity). The OrthoANI and dDDH values between strain Q4567T and C. morbi ATCC 51271T were respectively 79.43% and 23.8%. Therefore, we concluded that strain Marseille-Q4567T represents a novel species of the genus Catonella, for which the name Catonella massiliensis sp. nov. is proposed (= CSUR Q4567).

Isolation and Characterization of Kingella bonacorsii sp. nov., A Novel Kingella Species Detected in a Stable Periodontitis Subject.

Members of the genus Kingella are mostly commensals of the oral cavity, but some of them are involved in invasive infections, especially in young children. This study provides new knowledge on the diversity of this genus by describing a novel species of Kingella isolated from a dental plaque sample from a 51-year-old man with a history of periodontitis. Morphological and chemotaxonomic characteristic were investigated using different growth conditions, pH and temperature. Cellular fatty acid methyl ester (FAME) analysis was performed by gas chromatography/mass spectrometry (GC/MS). Phylogenetic analysis based on 16S rRNA, orthologous average nucleotide identity (OrthoANI) and digital DNA–DNA hybridization (dDDH) relatedness were also performed. Strain Marseille-Q4569T was found to be a facultative aerobic, nonmotile and non-spore-forming rod-shaped bacterium that grows at 28–41.5 °C (optimum 37 °C), pH 5.5–8.5 (optimum pH 7.5) and 5–15 g/L of NaCl. The major fatty acids were Hexadecanoic acid (32.7%), 11-Octadecenoic acid (26.1 %) and 9-Hexadecenoic acid (21.3 %). Despite high 16S rRNA gene sequence similarity (98.72%) between strain Marseille-Q4569T and Kingella oralis strain UB-38T, the degree of OrthoANI was at the limit of the cutoff (95.83%), and the degree of dDDH was lower (63.6%) than thresholds used to delineate prokaryotic species. Therefore, it is proposed that strain Marseille-Q4569T represents a novel species of the genus Kingella, for which the name Kingella bonacorsii sp. nov. is proposed (=CSUR Q4569).

Enterococcus saigonensis sp. nov., isolated from retail chicken meat and liver.

Two Gram-stain-positive strains, VE80T and VE116, which were resistant to vancomycin, were isolated from retail chicken meat and liver in Ho Chi Minh, Vietnam, respectively. These strains were characterized by sequence analyses of 16S rRNA, RNA polymerase α-subunit (rpoA), ATP synthase α-subunit (atpA), and phenylalanyl-tRNA synthase α-subunit (pheS) genes, determination of DNA G+C content, cellular fatty acid methyl ester analysis, DNA-DNA hybridization, and conventional morphological and biochemical tests. Strains VE80T and VE116 had 99.6 % 16S rRNA gene sequence similarity with Enterococcus canintestini LMG 13590T, and 99.1 % 16S rRNA gene sequence similarity with Enterococcus dispar ATCC 51266T. However, the two isolates could be clearly differentiated from these reference strains by the low sequence similarities (86.1-86.8 %) of the atpA gene, low DNA-DNA relatedness (<22.8 %), and differences in the production of acid from melezitose and methyl α-d-glucoside. Based on the results obtained in the present study, these two isolates are considered to represent a novel species of the genus Enterococcus, for which the name Enterococcus saigonensis sp. nov., is proposed. The type strain is VE80T (=JCM 31193T=CCUG 68827T).

Description of Kibdelosporangium banguiense sp. nov., a novel actinomycete isolated from soil of the forest of Pama, on the plateau of Bangui, Central African Republic.

A novel actinomycete strain F-240,109(T) from the MEDINA collection was isolated from a soil sample collected in the forest of Pama, on the plateau of Bangui, Central African Republic. The strain was identified according to its 16S rRNA gene sequence as a new member of the genus Kibdelosporangium, being closely related to Kibdelosporangium aridum subsp. aridum (98.6% sequence similarity), Kibledosporangium phytohabitans (98.3%), Kibdelosporangium aridum subsp. largum (97.7%), Kibdelosporangium philippinense (97.6%) and Kibledosporangium lantanae (96.9%). In order to resolve its precise taxonomic status, the strain was characterised through a polyphasic approach. The strain is a Gram-stain positive, aerobic, non-motile and catalase-positive actinomycete characterised by formation of extensively branched substrate mycelia and sparse brownish grey aerial mycelia with sporangium-like globular structures. The chemotaxonomic characterisation of strain F-240,109(T) corroborated its affiliation into the genus Kibdelosporangium. The peptidoglycan contains meso-diaminopimelic acid; the major menaquinone is MK-9(H4); the phospholipid profile contains high amounts of phosphatidylethanolamine, hydroxyphosphatidylethanolamine, diphosphatidylglycerol and an unidentified phospholipid; and the predominant cellular fatty acid methyl esters are iso-C16:0, iso-C14:0, iso-C15:0 and 2OH iso-C16:0. However, some key phenotypic differences regarding to its close relatives and DNA-DNA hybridization values indicate that strain F-240,109(T) represents a novel Kibdelosporangium species, for which the name Kibdelosporangium banguiense sp. nov. is proposed. The type strain is strain F-240,109(T) (=DSM 46670(T), =LMG 28181(T)).

Gas chromatography-mass spectrometry method for rapid identification and differentiation ofBurkholderia pseudomalleiandBurkholderia malleifrom each other,Burkholderia thailandensisand several members of theBurkholderia cepaciacomplex

To develop a simple gas chromatography-mass spectrometry (GC-MS) method for the detection and differentiation of Burkholderia pseudomallei and Burkholderia mallei from each other, Burkholderia thailandensis and several members of the Burkholderia cepacia complex. Biomarkers were generated by one-step thermochemolysis (TCM) and analysed using a GC-MS system. Fragments of poly-3-hydroxybutyrate-co-hydroxyvalerate [poly(3HBA-co-3HVA)] produced by TCM were useful biomarkers. Several cellular fatty acid methyl esters were important in differentiating the various Burkholderia species. A statistical discrimination algorithm was constructed using a combination of biomarkers. The identities of four B. pseudomallei strains, four B. mallei strains and one strain of each near neighbour were confirmed in a statistically designed test using the algorithm. The detection limit for this method was found to be approximately 4000 cells. The method is fast, accurate and easy to use. The algorithm is robust against different growth conditions (medium and temperature). This assay may prove beneficial in a clinical diagnostic setting, where the rapid identification of B. pseudomallei is essential to effective treatment. This method could also be easily employed after a biological attack to confirm the presence of either B. pseudomallei or B. mallei.

Oscillibacter ruminantium sp. nov., isolated from the rumen of Korean native cattle

A strictly anaerobic, Gram-negative, non-spore-forming bacterium, designated GH1(T), was isolated from the rumen of Korean native cattle (HanWoo). Cells were straight to slightly curved rods (2.0-4.5 µm long) and were motile by peritrichous flagella. The isolate grew at 30-45 °C (optimum 40 °C), at pH 5.5-6.5 (optimum pH 6.0) and with up to 3.5% (w/v) NaCl. Strain GH1(T) produced acid from d-glucose, d-ribose and d-xylose, with butyric acid being the major end product. The genomic DNA G+C content was 54.6 mol%. Based on comparative 16S rRNA gene sequence analysis, strain GH1(T) was most closely related to Oscillibacter valericigenes Sjm18-20(T) (97.3% 16S rRNA gene sequence similarity). DNA-DNA hybridization between strain GH1(T) and O. valericigenes DSM 18026(T) showed 24% reassociation. The major fatty acids were iso-C13:0 (13.0%), iso-C15:0 (17.6%), anteiso-C15:0 (8.4%) and C14:0 (4.1%), and the cellular fatty acid methyl esters as dimethylacetals (DMAs) were C16:0 DMA (17.8%), iso-C15:0 DMA (15.2%) and C14:0 DMA (4.52%). The cell-wall peptidoglycan of strain GH1(T) contained meso-diaminopimelic acid and the major cell-wall sugar was galactose. Based on 16S rRNA gene sequence similarity, phylogenetic analysis, DNA G+C content, DNA-DNA relatedness and distinct phenotypic characteristics, strain GH1(T) is classified in the genus Oscillibacter as a member of a novel species, for which the name Oscillibacter ruminantium sp. nov. is proposed. The type strain is GH1(T) (=KCTC 15176(T)=NBRC 108824(T)=JCM 18333(T)).

Using a different growth medium greatly improves distinction of Butyrivibrio fibrisolvens and Pseudobutyrivibrio xylanivorans strains by the cellular fatty acids and aldehydes profiles

A total of 11 ruminal strains currently assigned to Butyrivibrio fibrisolvens and Pseudobutyrivibrio xylanivorans were cultivated in two different media, rumen fluid containing M2 and short-chain fatty acid (SCFA) containing M330, and their cellular fatty acid methyl esters (FAME) and dimethylacetals (DMA) were analyzed using gas chromatography. A comparison of the FAME/DMA compositions revealed that the difference in SCFA contents in the growth medium induced a pronounced quantitative effect on the cellular branched-chain fatty acid and aldehydes proportions only in the P. xylanivorans strains. This study shows that FAME/DMA analysis is a powerful chemotaxonomic tool in the group of phenotypically similar rumen butyrivibria especially when the influence of the growth medium is evaluated.

Microorganisms participating in the biodegradation of modified polyethylene films in different soils under laboratory conditions

The degree of biodegradation of low-density polyethylene (LDPE) films modified with Bionolle ® polyester in different soils under laboratory conditions was evaluated. Films were incubated in soils from waste coal, a forest and an extinct volcano crater. Prior to degradation studies, soils underwent chemical and microbiological analysis. Film weight loss and mechanical properties, as well as the surface of the polymeric samples determined via scanning electron microscopy, were evaluated after 75, 150 and 225 days of biodegradation. Important chemical changes in the polymeric chains were detected by Fourier Transform Infrared Spectroscopy (FTIR). Fungal and bacterial species that were able to grow on the film surfaces were monitored in order to see whether the films were easily colonised by autochthonous microorganisms (i.e., typical to each soil). Identification of microorganisms was based on their cellular fatty acid methyl ester (FAME) profiles. Biodegradation of modified polyethylene films in soils led to significant changes (i.e., elongation at brake of 98%) in their mechanical properties that were caused by biochemical modifications of both polyester and polyethylene. Compared to waste coal soil, films underwent rapid biodegradation in soils that were rich in organic matter. Bacteria belonging to the genus, Bacillus, and the fungi, Gliocladium viride, Aspergillus awamori and Mortierella subtilissima, were easily able to colonise both polyethylene and polyethylene modified with Bionolle ®.

Using a different growth medium greatly improves distinction of Butyrivibrio fibrisolvens and Pseudobutyrivibrio xylanivorans strains by the cellular fatty acids and aldehydes profiles

A total of 11 ruminal strains currently assigned to Butyrivibrio fibrisolvens and Pseudobutyrivibrio xylanivorans were cultivated in two different media, rumen fluid containing M2 and short-chain fatty acid (SCFA) containing M330, and their cellular fatty acid methyl esters (FAME) and dimethylacetals (DMA) were analyzed using gas chromatography. A comparison of the FAME/DMA compositions revealed that the difference in SCFA contents in the growth medium induced a pronounced quantitative effect on the cellular branched-chain fatty acid and aldehydes proportions only in the P. xylanivorans strains. This study shows that FAME/DMA analysis is a powerful chemotaxonomic tool in the group of phenotypically similar rumen butyrivibria especially when the influence of the growth medium is evaluated.

Use of Fatty Acid Methyl Ester Profiles for Discrimination ofBacillus cereusT-Strain Spores Grown on Different Media

The goal of this study was to determine if cellular fatty acid methyl ester (FAME) profiling could be used to distinguish among spore samples from a single species (Bacillus cereus T strain) that were prepared on 10 different medium formulations. To analyze profile differences and identify FAME biomarkers diagnostic for the chemical constituents in each sporulation medium, a variety of statistical techniques were used, including nonmetric multidimensional scaling (nMDS), analysis of similarities (ANOSIM), and discriminant function analysis (DFA). The results showed that one FAME biomarker, oleic acid (18:1 omega9c), was exclusively associated with spores grown on Columbia agar supplemented with sheep blood and was indicative of blood supplements that were present in the sporulation medium. For spores grown in other formulations, multivariate comparisons across several FAME biomarkers were required to discern profile differences. Clustering patterns in nMDS plots and R values from ANOSIM revealed that dissimilarities among FAME profiles were most pronounced when spores grown with disparate sources of complex additives or protein supplements were compared (R > 0.8), although other factors also contributed to FAME differences. DFA indicated that differentiation could be maximized with a targeted subset of FAME variables, and the relative contributions of branched FAME biomarkers to group dissimilarities changed when different media were compared. When taken together, these analyses indicate that B. cereus spore samples grown in different media can be resolved with FAME profiling and that this may be a useful technique for providing intelligence about the production methods of Bacillus organisms in a forensic investigation.

Diversity and high nitrogenase activity of endophytic diazotrophs isolated from Oryza rufipogon Griff.

Diversity and nitrogenase activity of endophytic diazotrophs colonized in the wild rice Oryza rufipogon Griff grown in Boluo, Huilai County in Guangdong Province and Lingshui County in Hainan Province were studied. Thirty-seven isolates obtained from Oryza rufipogon were identified as putative endophytic nitrogen-fixing bacteria by ARA (acetylene reduction assay) test and further confirmed by PCR amplification of nifH gene fragments. All obtained strains have ARA activity and the same sized nifH gene fragments. Above the similarity level of 80%, the obtained isolates were assigned as Group I to VIII by the clustering of IS-PCR fingerprints. The SDS-PAGE whole-cell protein patterns were similar to those of IS-PCR fingerprints. Components and contents of fatty acid methyl esters (FAMEs) were used to differentiate the representative strains (Ls13, Ls8, BL1, BL12, HL6, Ls4) from Group I to Group VI. The six representative strains showed significant difference in contents and components of cellular fatty acid methyl ester. 16S rDNA sequencing analysis showed that strains of Group I to VII were located in Enterobacteraceae (γ-proteobacteria). Strains of Group I and Group II were closely related to Klebsiella sp.; Strain Ls8 of Group II was a little far away from the genus of Pantoea (homology level 96% with Pantoea agglomerans), which may represent a new species or genus in Enterobacteraceae; Strains of Groups IV and V belonged to different Enterobacter sp.; Strain Ls4 and Ls 9 representing Group VI were close to Citrobacter amalonaticus with 98% sequence similarity; Strain Ls15 of Group VII showed 98% sequence identity with Pantoea sp.; Strains of Group VIII were assigned to the genus Ideonella (β-proteobacteria). Based on the above results, endophytic diazotrophs isolated from O. rufipogon showed great diversity and some diazotrophs showed high nitrogenase activity with 42.52 μmol/mL·h C2H4. Inoculation to rice tests indicated that the isolated endophytic diazotrophs significantly promoted the rice growth.

A multiplex polymerase chain reaction assay for the identification of Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis

The objective of this study was to design a multiplex PCR assay to identify Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis. The multiplex PCR included primer sets HP, amplifying a DNA region from an unknown hypothetical protein, Lkt and Lkt2, amplifying different regions of the leukotoxinD gene, and 16S to amplify universal bacterial sequences of the 16S rRNA gene. Based on positive amplification, isolates were delineated as M. haemolytica (HP, Lkt, 16S), M. glucosida (HP, Lkt, Lkt2, 16S), or M. ruminalis (HP, 16S). The validity of the assay was examined against 22 reference strains within the family Pasteurellaceae and 17 field isolates (nasal) that had been collected previously from feedlot cattle and tentatively identified as M. haemolytica based on morphology and substrate utilization. Additionally, 200 feedlot cattle were screened for M. haemolytica using multiplex PCR. Forty-four isolates from 25 animals were identified as M. haemolytica. The PCR assay positively identified all M. haemolytica, as confirmed by phenotypic tests and clustering based upon cellular fatty acid methyl ester (FAME) profiles. Selected nasal isolates that exhibited evidence of haemolysis, but were M. haemolytica-negative based on PCR, were also confirmed negative by phenotypic and FAME analyses. The multiplex PCR assay required no additional phenotypic tests for confirmation of M. haemolytica, within the group of bacteria tested.

Evaluation of multiple strains of Enterobacter sakazakii using fatty acid profiles

Fatty acid profiles are useful for identifying Gram-negative Enterobacter sakazakii strains within the family Enterobacteriaceae. The majority of cases of E. sakazakii infection have involved sepsis, meningitis, or enteritis, especially in neonates and infants. Gas chromatography with flame ionization detection (GC-FID) was utilized for the analysis of cellular fatty acid methyl esters (FAMEs). Thirty E. sakazakii strains isolated from food and environmental sources were cultured for 24 h on brain heart infusion (BHI) agar on three different days at 37 °C. Whole cell FAMEs were obtained by saponification, methylation and extraction into hexane:methyl tert-butyl ether. The day to day variations for the 30 E. sakazakii strains for each fatty acid were determined. Gram-negative bacteria commonly contain combinations of straight-chain, unsaturated, hydroxyl, and cyclo fatty acids. Major fatty acids of E. sakazakii strains evaluated in this study were straight chain 12:0, 14:0, 16:0 and unsaturated 16:1, 18:1, and 17:0 ω cyclo 7–8. Analysis of FAMEs from E. sakazakii strains grown on BHI agar by this rapid GC-FID method is highly reproducible and provides a sensitive procedure for identification of this organism. The fatty acid profile assay could be used to rapidly screen infant formula samples for E. sakazakii and reduce the time required for the current assay by up to 5 days.

Differentiation of Species Combined into the Burkholderia cepacia Complex and Related Taxa on the Basis of Their Fatty Acid Patterns

Using the established commercial system Sherlock (MIDI, Inc.), cellular fatty acid methyl ester analysis for differentiation among Burkholderia cepacia complex species was proven. The identification key based on the diagnostic fatty acids is able to discern phenotypically related Ralstonia pickettii and Pandoraea spp. and further distinguish Burkholderia pyrrocinia, Burkholderia ambifaria, and Burkholderia vietnamiensis.

Identification of foodborne bacteria by infrared spectroscopy using cellular fatty acid methyl esters

Identification of bacterial species by profiling fatty acid methyl esters (FAMEs) has commonly been carried out by using a 20-min capillary gas chromatographic procedure followed by library matching of FAME profiles using commercial MIDI databases and proprietary pattern recognition software. Fast GC (5 min) FAME procedures and mass spectrometric methodologies that require no lipid separation have also been reported. In this study, bacterial identification based on the rapid (2 min) infrared measurement of FAME mixtures was demonstrated. The microorganisms investigated included Gram positive bacteria Staphylococcus aureus, Listeria monocytogenes, Bacillus anthracis, and Bacillus cereus, and Gram negative bacteria from the family Enterobacteriacae: Yersinia enterocolitica, Salmonella typhimurium, Shigella sonnei, and Escherichia coli (four strains of E. coli), and non-Enterobacteriacae: Vibrio cholerae, Vibrio vulnificus, and Vibrio parahemolyticus. Foodborne bacterial mixtures of FAMEs were measured by using an attenuated total reflection (ATR)–Fourier transform infrared (FTIR) spectroscopic procedure and discriminated by multivariate analysis. Results showed that the Enterobacteriacae could be discriminated from the vibrios. The identification was at the level of species (for the Bacillus and Vibrio genera) or strains (for the E. coli species). A series of bacterial FAME test samples were prepared and analyzed for accuracy of identification, and all were correctly identified. Our results suggest that this infrared strategy could be used to identify foodborne pathogens.

Species-Specific Identification of Commercial Probiotic Strains

Products containing probiotic bacteria are gaining popularity, increasing the importance of their accurate speciation. Unfortunately, studies have suggested that improper labeling of probiotic species is common in commercial products. Species identification of a bank of commercial probiotic strains was attempted using partial 16S rDNA sequencing, carbohydrate fermentation analysis, and cellular fatty acid methyl ester analysis. Results from partial 16S rDNA sequencing indicated discrepancies between species designations for 26 out of 58 strains tested, including two ATCC Lactobacillus strains. When considering only the commercial strains obtained directly from the manufacturers, 14 of 29 strains carried species designations different from those obtained by partial 16S rDNA sequencing. Strains from six commercial products were species not listed on the label. The discrepancies mainly occurred in Lactobacillus acidophilus and Lactobacillus casei groups. Carbohydrate fermentation analysis was not sensitive enough to identify species within the L. acidophilus group. Fatty acid methyl ester analysis was found to be variable and inaccurate and is not recommended to identify probiotic lactobacilli.

Bacterial Populations Associated with Rice Seed in the Tropical Environment

ABSTRACT During the 1995 wet season, harvested rice seed was collected from farmers' fields at different locations in Iloilo, Philippines. Bacterial isolations from crushed seed yielded 428 isolates. The isolates were characterized by BOX-polymerase chain reaction fingerprinting of total genomic DNA and represented 151 fingerprint types (FPT). Most FPTs were found on a single occasion, although matching fingerprints for isolates from different samples also were found. Identifications were made by cellular fatty acid methyl ester analysis and additional use of Biolog GN/GP MicroPlates and API 20E/50CHE systems. The predominant bacteria were Enterobacteriaceae (25%), Bacillus spp. (22%), and Pseu-domonas spp. (14%). Other bacteria regularly present were identified as Xanthomonas spp., Cellulomonas flavigena, and Clavibacter michiganense. Of the total number of isolated bacteria, 4% exhibited in vitro antifungal activity against Rhizoctonia solani or Pyricularia grisea. Two percent of isolates were pathogens identified as Burkholderia glumae and Burkholderia gladioli. Five percent of isolates induced sheath necrosis on only 50 to 90% of inoculated plants and were related to Bacillus pumilus, Paenibacillus spp., Pseudomonas spp., and Pantoea spp.

Comparison of the Antimicrobial Tolerance of Oxytetracycline-Resistant Heterotrophic Bacteria Isolated from Hospital Sewage and Freshwater Fishfarm Water in Belgium

The aim of this study was to investigate the relationship between antimicrobial tolerance and taxonomic diversity among the culturable oxytetracycline-resistant (Ot(r)) heterotrophic bacterial population in two Belgian aquatic sites receiving wastewater either from human medicine or from aquaculture. The study of Ot(r) heterotrophs and mesophilic Aeromonas spp. allowed comparison of tolerance data at the intergenus as well as at the intragenus level. In total, 354 independently obtained Ot(r) isolates were subjected to antimicrobial tolerance testing and identified by GLC analysis of their cellular fatty acid methyl esters (FAMEs), by API 20E profiling and/or by Fluorescent Amplified Fragment Length Polymorphism (FAFLP) DNA fingerprinting. In general, Ot(r) hospital heterotrophs displayed a higher frequency (84%) of ampicillin (Amp) tolerance compared to the Ot(r) heterotrophs from the freshwater fishfarm site (22%). FAME results indicated that this effect was linked to the predominance of intrinsically ampicillin-resistant Ot(r) Aeromonas strains over representatives of Acinetobacter and Escherichia coli within the hospital strain set. Among the Ot(r) mesophilic Aeromonas strain set, the global tolerance profiles of the two sites only differed in a higher number of kanamycin (Kan) -tolerant strains (43%) for hospital aeromonads in comparison with the fishfarm aeromonads (8%). To some extent, this finding was correlated with the specific presence of Aeromonas caviae DNA hybridisation group (HG) 4. Collectively, these results suggest that the profiles for Amp and Kan tolerance observed in both sites arose from taxonomic differences in the culturable Ot(r) bacterial population at the generic or subgeneric level. In addition, our identification data also revealed that Enterobacter sp., Stenotrophomonas maltophilia, and A. veronii biovar sobria HG8 may be considered potential indicator organisms to assess microbial tolerance in various compartments of the aquatic environment.